Mitochondrial nicotinamide nucleotide transhydrogenase (TH) is a homodimer of monomer M(r)=109228, is located in the inner membrane, and catalyzes hydride ion transfer between the 4A position of NAD(H) and the 4B position of NADP(H) in a reaction that is coupled to transmembrane proton translocation with a H+/H- stoichiometry close to unity. Among other things, the matrix NADPH so produced is utilized by the combined actions of glutathione reductase and glutathione peroxidase to consume H2 0 2, which results from dismutation of O2- generated by the respiratory chain. H2O2 and hydroxyl radicals produced via the Haber-Weiss and Fenton reactions can initiate lipid peroxidation and cause extensive damage to mitochondrial membranes and mtDNA. During the past grant period, we have deduced the amino acid sequences of the mature TH (1043 residues) and its signal peptide (43 residues); identified the NAD and NADP binding domains and the DCCD and N-ethylmaleimide (NEM) binding sites; investigated the membrane topography of TH; shown that NADP and NADPH, but not NAD and NADH, binding alters TH conformation in different ways; and arrived from these and previous studies at a working hypothesis regarding the mechanism of energy transduction and H+ translocation by TH. This mechanism involves utilization of substrate binding energy for proton translocation via TH conformation change. Studies contemplated for the next grant period include: I. Structural Studies: (a) Further study of the topography of the central hydrophobic domain of TH. (b) Study of the spatial relationship between the NAD- and NADP-binding domains, which are, respectively, the N- and C-terminal extramembranous segments of TH. (c) Further elucidation of the NAD(H) binding site. II. Mechanistic Studies: (a) Study of the effect of membrane potential on Kd for NADP and NADPH, and on TH conformation change as monitored by the rate of TH modification by various proteases and NEM. (b) Reconstitution of bovine TH lacking the N-terminal NAD(H)-binding extramembranous segment (43 kDa) and mutated E. coli membranes lacking the TH alpha subunit (corresponds to bovine 43 kDa plus a short hydrophobic tail) with purified, soluble 43 kDa and possibly other 4A-specific NAD-binding proteins. (c) Proteinase K treatment of mitoplasts splits TH into a 72 kDa N-terminal and a 37 kDa C-terminal fragment. The bisected TH has been purified and shown to have high TH activity and H+ translocation capability when embedded in liposomes. Attempt will be made to separate the 37 kDa fragment and reconstitute it with the soluble 43 kDa piece and demonstrate H+ translocation. If successful, these studies will confine the TH machinery for utilization of substrate binding energy for H+ translocation to the C-terminal 37 kDa fragment, which contains a short hydrophobic head capable of forming only 5 membrane-intercalating amino acid clusters. III. E. Coli TH will be isolated, its NAD and NADP binding sites will be determined, its possible substrate-promoted conformation change will be investigated, and attempts will be made to crystallize it.